UNIQUE ADVANTAGES OF USING LOW-TEMPERATURE SCANNING ELECTRON-MICROSCOPY TO OBSERVE BACTERIA

Electron microscopy (EM) has greatly helped to elucidate our understan ding of bacterial structure and function. However, several recent stud ies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems , the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leav es (Spinacia oleracea L. cv. New Jersey), which were naturally infecte d or inoculated with bacteria, were used as the experimental material. I cm segments of the infected leaves were plunge frozen in liquid nit rogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hy drated leaf segments were transferred onto agar medium to determine wh ether preparation for LTSEM was nondestructive to the bacteria. The ot her tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LT SEM: (i) viable bacteria, which were recovered from the leaf sample, c ould be cultured on agar medium for subsequent study, and (ii) the fro zen samples could be freeze substituted and embedded so that transmiss ion electron microscopic (TEM) observations could be carried out on th e same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either proce ssed for TEM observation to obtain further structural details or recov ered to culture the pathogenic bacteria for supplementary studies.